Throughout this application various publications are cited within parenthesis. Full bibliographic citations for these references may be found listed alphabetically at the end of the specification immediately preceding the claims. The disclosures for these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
The present invention relates generally to the control of pathogens in plants and animals and more particularly to the control of viruses and viroids in transgenic plants. Further, it relates to the control of viral pathogens in mammals.
The ability to control plant pathogens has long been a principal goal in agricultural research. In contemporary research, there has been a focus on recombinant DNA technology in the quest for developing disease-resistant plants. However, despite the commercially devastating effects of various diseases in, for example, crop plants, little progress has been made in controlling viroid and virus infection in plants.
A viroid is a plant-pathogenic infectious agent comprising a naked (i.e. non-protein associated) circular single stranded RNA molecule. Most known viroids are similar in structure and apparently rely solely on host cell enzymes for replication. Replication is thought to occur in the host cell nucleus where the RNA is associated with the nucleolus. Infectious viroid RNA (+) is transcribed into an oligomeric complementary RNA (xe2x88x92) several times the unit length of the viroid (+) RNA. The (xe2x88x92) RNA oligomers probably then serve as templates for the synthesis of (+) RNA which is then cleaved into unit-length linear strands followed by ligation to form single stranded infectious (+) RNA molecules.
The viroid class of plant pathogens are low molecular weight, circular, single-stranded RNA molecules. They are the smallest known autonomously replicating agents with a size range between 246-375 nucleotides. The RNA has been shown in have extensive intramolecular base-pairing giving the viroids a rod-like structure (Sanger et al., 1976). The result of the interaction of the viroids with the plant host can range from no apparent or a mild associated host phenotype to an extremely severe pathology. In many cases the infection can lead to significant impairment of the plant growth and fruiting causing economically important disease. Although the pathology of viroid infections has been well documents, there has been no report of the identification of resistance genes of the design and introduction of synthetic resistance genes into host plants. A contributing factor to this is that details of the mechanisms and onset of pathogenesis are not well understood. As such, it is difficult to identify host gene targets to breed for a resistant genotype. A potentially useful approach to bypass this problem and to lead to the development of viroid-resistant cultivars is the application of the concept of pathogen-derived resistance mechanisms (Sanford and Johnston, 1985).
Pathogen-derived resistance includes strategies which involve the expression of a component of the pathogen in transgenic hosts resulting in increased tolerance or resistance in the host when infected with that pathogen. Pathogen-derived resistance has been applied to a wide range of plant virus and host combinations. Some of the successful methods involving the expression of virus open reading frames (ORF) in transgenic plant hosts include the expression of virus capsid protein genes (for review see Beachy et al., 1990) and, in the case of Tobacco Mosaic Virus and Pea Early Browning Virus, the expression of the readthrough sequence of the virus-encoded RNA polymerase gene (Golemboski et al., 1990, Macfarlane and Davies, 1992). Although very effective against viruses, these techniques are unsuitable for application to viroids as they do not have identifiable ORFs (Sanger, 1987). However, the fact that the viroids are single-stranded RNA molecules makes them potentially sensitive to resistance strategies involving the expression of transgenes encoding either antisense or ribozyme RNA molecules. These gene manipulation techniques that result in regulation most likely via a primary interaction between the transgene transcript and a target RNA molecule have been shown to be extremely effective in plants. Antisense-mediated control of gene expression has been demonstrated for endogenous plant genes in the case of inhibition of a number of endogenous genes. In addition, antisense strategies have been successful in conferring a degree of resistance to the viral pathogens potato virus X; cucumber mosaic virus and potato leafroll virus (Hemenway et al., 1988, Cuozzo et al., 1988; Kawchuk et al., 1991) in the appropriate plant hosts. Ribozyme-mediated gene inactivation strategies have been employed against the bacterial neomycin phosphotransferase gene in protoplasts of Nicotiana tabacum (Steinecke et al., 1992) and TMV replication in protoplasts (Edington and Nelson, 1992). As yet, neither technique has been evaluated against the viroid class of pathogens.
This application describes the first reported test of these strategies to engineer resistance to viroids. The experimental system involved the well characterized Citrus Exocortis Viroid and the readily transformable tomato host, Lycopersicon lycopersicum cv. UC82B. The combination of the CEV Australian isolate (CEV A, Visvader et al., 1982) and the Lycopersicon lycopersicum UC82B cultivar was chosen as the productive infection. Although this viroid and host combination results in viroid replication to the same level as infection of the symptom-showing host L. lycopersicum Mill cv. Rutgers, the interaction produces negligible symptoms and allows assessment of viroid replication independently of the devastating symptoms seen in the latter cultivar. The strategies which were tested involved the expression of transgenes encoding antisense and long ribozyme genes targeting either the viroid-sense (positive strand) RNA molecule or the complementary (negative strand) viroid RNA. The rationale for the selection of two target RNAs was that it is possible that the positive and negative strand RNAs may represent quite different targets in terms of abundance, cellular localization and/or structure.
Although the full details of the viroid replication cycle have not been elucidated it is generally accepted that the replication cycle involves the synthesis of the negative strand template. From this negative strand, copies of positive strand molecules are formed which are subsequently processed into circular viroid molecules (Symons, 1990). For CEV it has been reported that the negative strand is much less abundant than the positive strand in infected plants and that the distribution of RNA, in terms of multimeric units, is different for the two RNA species (Hutchins et al., 1985).
Although the exact cause of the pathogenicity of viroids is yet to be established, it is possible that the viroids interfere with pre-RNA processing in host cells. The result in an array of symptoms ranging from mild (e.g. discoloration and malformation of leaves) to severe and lethal.
There is a need, therefore, to develop a method for effectively controlling infection of viroids, viruses, and other viroid-like infectious agents in plants. In addition, there is a need for methods for controlling virus pathogens of animals.
The invention consists of a non-naturally occurring nucleic acid molecule capable of blocking or interfering with a replicative intermediate of a virus, a virusoid, or a viroid. The nucleic acid molecule may contain a ribozyme or a plurality of ribozymes. Alternatively, the nucleic acid molecule may be an antisense nucleic acid molecule. The ribozyme may be a hairpin ribozyme, a hammerhead ribozyme, an RNAase P ribozyme, a minizyme, or other catalytic RNA molecule.
The virus may be an animal, a mammalian, a plant, a fungal, a protozoan, a yeast, a bacterial virus, or a human virus. The nucleic acid molecule may be expressed in the cell or it may be preformed and administered ex vivo.
The present invention contemplates a methods of controlling infection of a pathogenic infectious agent in a plant or animal.